RESUMO
INTRODUCTION: Fat cell differentiation (FCD) potentiates adipose cell characteristics including lipid storage and insulin sensitivity. In vitro, we have demonstrated that CCN2, also known as connective tissue growth factor (CTGF), inhibits FCD in NIH3T3-L1 cells and in adipocytes isolated from mouse epididymal fat pads. The aim of this study was to determine if the CCN2 effect on FCD is dependent on TGF-ß and TGF-ß downstream pathway signalling. METHODS: NIH3T3-L1 cells were differentiated using standard methods with IBMX/Dex/Insulin. FCD at day 10 was confirmed by induced gene markers resistin and adiponectin and by lipid accumulation. Cells were treated at d0 with single dose active rhTGF-ß1 (2 ng/mL), rhCCN2 (500 ng/mL) and/or TGF-ß type 1 receptor blocker (SB431542, 5 µM). Early induction of FCD transcription factors: CCAAT/enhancer binding proteins (C/EBPs) and peroxisome proliferator-activated receptor-γ (PPAR-γ), were also determined. RESULTS: In an early time course from 2 h, single doses of rhTGF-ß1 or rhCCN2 significantly inhibited by ~70 % the induction of C/EBP-ß and -δ mRNA, and also nuclear protein levels otherwise seen during FCD, whereas only delayed effects on PPAR-γ, at 48 h, occurred. Furthermore, the CCN2 inhibition of FCD markers adiponectin and resistin and lipid accumulation by Oil red O stain were each prevented by TGF-ß receptor blockade. Similar prevention was found using pan-specific anti-TGF-ß neutralising antibody. CCN2 and TGF-ß treatment each rapidly phosphorylated SMAD-3 signalling in early stages of FCD. CONCLUSION: This work shows novel findings that CCN2 effects on FCD are both TGF-ß and TGF-ß pathway dependent and are related to early effects on C/EBPs.
RESUMO
Adipocyte differentiation is a key process implicated in the pathogenesis of obesity and insulin resistance. Its regulation is triggered by a cascade of transcription factors, including the CCAAT/enhancer binding proteins (C/EBPs) and peroxisome proliferator-activated receptor-gamma (PPARgamma). Growth factors such as transforming growth factor-beta1 (TGF-beta1) are known to inhibit adipocyte differentiation in vitro, via the C/EBP pathway, and in vivo, but whether a downstream mediator of TGF-beta1, connective tissue growth factor (CTGF), also known as CCN2, has a similar role is unknown. Mouse 3T3-L1 cells were differentiated into adipocytes by using standard methods, and effects and regulation of CTGF were studied. Intervention with recombinant human CTGF during differing stages of differentiation caused an inhibition in the development of the adipocyte phenotype, according to the gene expression of the differentiation markers adiponectin and PPARgamma, as well as suppression of lipid accumulation and expression of the lipogenic enzyme glycerol-3-phosphate dehydrogenase. Whereas CTGF gene expression promptly fell by 90% as 3T3-L1 preadipocytes differentiated into mature adipocytes, CTGF mRNA expression was induced by added TGF-beta1. CTGF applied to cells early in the course of differentiation inhibited total cell protein levels and nuclear localization of the beta-isoform of C/EBP (C/EBP-beta) and, subsequently, total cell C/EBP-alpha levels. CTGF also inhibited the adipocyte differentiation program in primary cultures of mouse preadipocytes. Expression of CTGF mRNA was twofold higher in the central fat depots of mice compared with subcutaneous fat, suggesting a potential role for CTGF in vivo. In summary, these data show that CTGF inhibits the adipocyte differentiation program.
